Project Summary Objective response rates to single agent checkpoint blockade immunotherapy (CBI) in recurrent or metastatic Head and Neck Squamous Cell Carcinoma (HNSCC) are low, in the range of 13-26%. Thus there is a critical need to develop combinatorial strategies to improve response rates and understand mechanisms of resistance. Our group has pioneered combinations of radiation (XRT) and CBI and demonstrated that XRT can enhance development of anti-tumor T-cell responses. However, while most studies have focused on T- cells, the second arm of the adaptive immune system, namely B-cell mediated anti-tumor antibody responses, has remained understudied. Anti-tumor antibodies play crucial roles in identifying and tagging tumor cells for destruction, activating immune responses, as well as limiting spread of viral infections. The primary goals of this project are to establish the role of B-cell mediated antibody responses in HNSCC and exploit mechanisms by which human papilloma virus (HPV) blocks immune responses. In Specific Aim 1 we will characterize the effects of XRT and XRT + CBI on the development and function of B-cell mediated anti- tumor antibody responses in HNSCC. We have identified that XRT and XRT+CBI can enhance development of B-cell IgG antibodies which contribute to tumor control. We will test the hypotheses that B-cell mediated antibody responses are critical for tumor control and that XRT enhances B-cell immune responses via increased antigen trafficking to the draining lymph node. We have discovered a novel HPV mediated resistance mechanism to CBI, whereby HPV E5 inhibits adaptive immune responses by blocking antigen processing and antigen presentation. In specific Aim 2 we will dissect this novel mechanism of HPV E5 mediated immune suppression and determine whether HPV E5 inhibitors enhance B-cell responses to XRT and CBI in HNSCC. Furthermore, we have identified that the antiviral drug and HPV E5 inhibitor Rimantadine has potent and novel anti-tumor effects in HNSCC. We will test the hypothesis that inhibition of HPV E5 improves response rates to CBI and XRT by restoring antigen processing and presentation. Our group is leading multiple clinical trials evaluating the efficacy of XRT and CBI in HNSCC and our preliminary data has identified that patients receiving XRT + CBI develop B-cell responses that correlate with response rates. In specific aim 3 we will establish whether increases in B-cell mediated IgG antibody responses correlate with objective response rates and overall survival in HNSCC patients. Completion of these specific aims will: 1) significantly improve our understanding of the immunological effects of XRT + CBI on B-cell anti-tumor immune responses in HNSCC; 2) elucidate a novel mechanism of HPV E5 mediated resistance to CBI and develop Rimantadine as a novel anti-cancer drug in HNSCC; and 3) establish whether B-Cell mediated IgG responses are a novel biomarker which correlate with responses and survival in HNSCC. Ultimately, these findings can be directly translated to improve outcomes for HNSCC patients.